Proteases play an important role in the regulation of many biological processes. They also play a major role in disease. In particular, proteolysis of primary polypeptide precursors is essential to the replication of several infectious viruses, including HIV and HCV. These viruses encode proteins that are initially synthesized as large polyprotein precursors Those precursors are ultimately processed by the viral protease to mature viral proteins. In light of this, researchers have begun to concentrate on inhibition of viral proteases as a potential treatment for certain viral diseases.
Proteases also play a role in non-infectious diseases. For example, changes in normal cellular function may cause an undesirable increase or decrease in proteolytic activity. This often leads to a disease state.
The ability to detect viral or mutant protease activity in a quick and simple assay is important in the biochemical characterization of these proteases and in the screening and identification of potential inhibitors. Several of these assays have been described in the art.
T. M. Block et al., Antimicrob. Agents Chemother., 34, pp. 2337-41 (1990) described a prototype assay for screening potential HIV protease inhibitors. This assay involved cloning the HIV protease recognition sequence into the tetracycline resistance gene (Tet.sup.R) of pBR322 and cotransfroming E. coli with the modified Tet.sup.R gene and the gene encoding the HIV protease. Coexpression of these two genes caused tetracycline sensitivity. Potential inhibitors were identified by the ability to restore tetracycline resistance to the transformed bacteria.
E. Sarubbi et al., FEBS Lett., 279, pp. 265-69 (1991) described another assay for detecting HIV protease inhibitors that utilized a HIV-1 Gag-.beta.-galactosidase fusion protein and a monoclonal antibody that bound to the fusion protein in the gag region. Coexpression of the HIV protease and the fusion protein lead to cleavage of the latter and abolished monoclonal antibody binding. Potential inhibitors were identified by increased binding of the monoclonal antibody to the fusion protein.
T. A. Smith et al., Proc. Natl. Acad. Sci. USA, 88, pp. 5159-62 (1991), B. Dasmahapatra et al., Proc. Natl. Acad. Sci. USA, 89, pp. 4159-62 (1992) and M. G. Murray et al., Gene, 134, pp. 123-28 (1993) each described protease assay systems utilizing the yeast GAL4 protein. Each of these authors described inserting a protease cleavage site in between the DNA binding domain and the transcriptional activating domain of GAL4. Cleavage of that site by a coexpressed protease renders GAL4 transcriptionally inactive leading to the inability of the transformed yeast to metabolize galactose.
H.-D. Liebig et al., Proc. Natl. Acad. Sci. USA, 88, pp. 5979-83 (1991) disclosed the use of a fusion protein consisting of a self-cleaving protease fused to the a fragment of .beta.-galactosidase to assay protease activity. Active forms of the protease cleaved themselves off of the fusion protein and the resulting protein was able to carry out .alpha.-complementation. Fusions containing inactive protease were unable to perform .alpha.-complementation.
Y. Komoda et al., J. Virol., 68, pp. 7351-57 (1994) described an assay to identify HCV protease cleavage sites within the HCV precursor polyprotein. These authors created chimeric proteins comprising various portions of the HCV precursor polyprotein inserted in between the E. coli maltose binding protein and dihydrofolate reductase. If the HCV portion of these chimeras contained a cleavage site, the chimera would be cleaved when it was coexpressed with HCV protease in E. coli. Cleavage of the chimera was determined by SDS-polyacrylamide gel electrophoresis of E. coli lysates.
Y. Hirowatari et al., Anal. Biochem., 225, pp. 113-120 (1995) described another assay to detect HCV protease activity. In this assay, the substrate, HCV protease and a reporter gene are cotransfected into COS cells. The substrate is a fusion protein consisting of (HCV NS2)-(DHFR)-(HCV NS3 cleavage site)-Tax1. The reporter gene is chloramphenicol transferase (CAT) under control of the HTLV-1 long terminal repeat (LTR) and resides in the cell nucleus following expression. The uncleaved substrate is expressed as a membrane-bound protein on the surface of the endoplasmic reticulum due to the HCV NS2 portion. Upon cleavage, the released Tax1 protein translocates to the nucleus and activates CAT expression by binding to the HTLV-1 LTR. Protease activity is determined by measuring CAT activity in a cell lysate.
Despite these developments, no one has yet developed a protease assay system that can be carried out with higher eukaryotic cells and is both quantitative and does not require cell lysis prior to quantitation. Avoiding cell lysis prior to quantitation is desirable in that the assay may be performed more rapidly and with less manipulation. Also, lysis can often lead to aberrant results. Thus, there is a need for an accurate and quantitative cellular-based protease assay that can be carried out in a higher eukaryotic cell without cell lysis.